Structurally Compact, Blue-Green-Red Fluorescence Trackers for the Outer Cell Membrane: Zwitterionic (Naphthylvinyl)pyridinium Dyes.

The cell membrane regulates the flux of materials in and out of cell, cell adhesion, and signaling. Fluorophores that selectively localize on it are in demand for investigations of the molecular events occurring on the outer cell membrane. Commercial membrane trackers based on phospholipids are structurally complex and difficult to modify further. We disclose the zwitterionic (naphthylvinyl)pyridinium dyes that selectively localize on the outer cell membrane and emit blue, green, and red fluorescence, respectively. Notably, they are structurally compact and provide bright fluorescence images of the cell membrane. By comparing with control compounds, we identified minimal structural elements for the "robust" localization of dye on the outer cell membrane. Further, the dyes are two-photon active, enabling high-resolution, deep-tissue imaging. One of the dyes was used to image a spleen tissue, which provided high-resolution fluorescent images with a distinct morphology. In addition, the materials and results disclosed are valuable for the development of membrane-targeting probes and structurally compact fluorophores.

[2 + 2] Photodimerization of Naphthylvinylpyridines through Cation-π Interactions in Acidic Solution.

Irradiation of (E)-4-(2-(2-naphthyl)vinyl)pyridine (1a) and (E)-4-(2-(1-naphthyl)vinyl)pyridine (1b) with a 250 W high-pressure mercury lamp in acidic solution afforded synHT dimers in high stereoselectivities. Similar results were obtained by visible light irradiation. On the other hand, when the reactions were carried out under neutral conditions, the stereoselectivities were very low, and the yields were decreased by visible light irradiation. Comparison of the UV-vis spectra between the acidic and the neutral conditions elucidated that the red shift was observed in acidic solutions. These results show that HCl plays essential roles not only in the preorientation of substrates through cation-π interactions, but also in the changes in the absorption properties of substrates that enable visible light reactions.

Novel naphthylstyryl-pyridium potentiometric dyes offer advantages for neural network analysis.

The submucous plexus of the guinea pig intestine is a quasi-two-dimensional mammalian neural network that is particularly amenable to study using multiple site optical recording of transmembrane voltage (MSORTV) [Biol. Bull. 183 (1992) 344; J. Neurosci. 19 (1999) 3073]. For several years the potentiometric dye of choice for monitoring the electrical activity of its individual neurons has been di-8-ANEPPS [Neuron 9 (1992) 393], a naphthylstyryl-pyridinium dye with a propylsulfonate headgroup that provides relatively large fluorescence changes during action potentials and synaptic potentials. Limitations to the use of this dye, however, have been its phototoxicity and its low water solubility which requires the presence of DMSO and Pluronic F-127 in the staining solution. In searching for less toxic and more soluble dyes exhibiting larger fluorescence signals, we first tried the dienylstyryl-pyridinium dye RH795 [J. Neurosci. 14 (1994) 2545] which is highly soluble in water. This dye yielded relatively large signals, but it was internalized quickly by the submucosal neurons resulting in rapid degradation of the signal-to-noise ratio. We decided to synthesize a series of naphthylstyryl-pyridinium dyes (di-n-ANEPPDHQ) having the same chromophore as di-8-ANEPPS and the quaternary ammonium headgroup (DHQ) of RH795 (resulting in two positive charges versus the neutral propylsulfonate-ring nitrogen combination), and we tested the di-methyl (JPW3039), di-ethyl (JPW2081), di-propyl (JPW3031), di-butyl (JPW5029), and di-octyl (JPW5037) analogues, all of them soluble in ethanol. We found that the di-propyl (di-3-ANEPPDHQ) and the di-butyl (di-4-ANEPPDHQ) forms yielded the best combination of signal-to-noise ratio, moderate phototoxicity and absence of dye internalization.

Molecular modeling of (E)-1-alkyl-4(3)-[2-(1H-azolyl)vinyl]-pyridinium salts and evaluation of their behavior towards choline acetyltransferase.

A new type of extended pi-system aza-analogue of (E)-4-[2-(1-naphthylvinyl)]-1-substituted pyridinium salts (NVP+) has been designed and its inhibitory activity towards choline acetyltransferase (ChAT) has been evaluated in vitro. Among the several examples of the title quaternary salts synthesized 2 and 3, the indolylvinylpyridinium salt 2e is the only one to show a very low ChAT inhibition. The molecular modeling study is highly illustrative of their behavior towards ChAT and interaction with the recognition site. Thus, several selected cations together with the reference NVP+ compound 1a were studied at the PM3 and AM1 levels respectively. At the global minima, all the compounds are planar, which, from the electron charge distribution, shows a degree of polarization similar to the NVP+ model compound 1a. However, the fitting of all optimized structures indicated that only the indole derivative 2e showed the same aromatic fragment orientation as 1a, which allows us to define a volume that is not accessible to ligands in the enzyme and consequently to a refined model of the choline acetyltransferase recognition site.

Molecular modeling studies of some choline acetyltransferase inhibitors.

Choline acetyltransferase (ChAT) inhibitors related to trans-1-methyl-4- (1-naphthylvinyl)-pyridinium (NVP+) have been assumed to depend upon a nearly or completely planar conformation for their enzyme-inhibitor interaction. In an effort to investigate the geometries and preferred conformations for these compounds, geometry optimizations using the semiempirical molecular orbital method AM1 and a modified version of the molecular mechanics method MM2(92) have been carried out. The results indicate that the active inhibitors are either planar or nearly coplanar, lying in relatively flat potential wells in the vicinity of the corresponding planar structures. When nonplanarity is favored, one ring is twisted out of the plane by approximately 30 degrees. Where steric features significantly deter assumption of a nearly planar conformation, the analogs are inactive. The inactivity of analogs bearing tricyclic aryl groups appears to result from bulk-related hindrance to ChAT receptor binding rather than lack of coplanarity.

Inhibition of brain choline acetyltransferase in vivo: (E)-1-methyl-4-(1-naphthylvinyl)-1,2,3,6-tetrahydropyridine hydrochloride (B115), a depot form of a potent inhibitor.

The quaternary ammonium salt (E)-4-(1-naphthylvinyl)pyridine hydroxyethyl bromide (B111) and the tertiary amine salt (E)-1-methyl-4-(1-naphthylvinyl)-1,2,3,6-tetrahydropyridine hydrochloride (B115), both previously shown to protect against organophosphate (OP) toxicity, were examined in vivo for effects on rat brain choline acetyltransferase (CAT) activity and acetylcholine (ACh) levels. When administered iv, but not when given ip, B111 was able to inhibit brain CAT 29% and reduce brain ACh levels 25%, yet was unable to prevent soman-induced increases in ACh. B115, which may serve as a depot form of a quaternary ammonium analogue, was able to decrease CAT activity as much as 80% upon multiple ip administration. This CAT inhibitory potency was unprecedented for a tertiary amine salt of its structure. However, ACh levels were reduced by no more than 25% and B115 was ineffective in preventing soman- and sarin-induced increases in ACh. Since the degree of inhibition of CAT activity produced by B111 and B115 was not accompanied by a corresponding decrease in ACh levels, the protection afforded by these compounds against OP toxicity is most likely not related to CAT inhibition. B115 was also tested for its ability to affect cholinergic receptor binding. B115 was administered to rats ip, twice daily, at low doses throughout a 3-week period. Analysis of cortex tissue revealed a 45% increase in nicotinic receptor binding with no change in either total muscarinic receptor binding (M-1 and M-2) or high-affinity muscarinic receptor binding (M-2 alone).

Synaptosomal "membrane-bound" choline acetyltransferase is most sensitive to inhibition by choline mustard.

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 2-(4-phenylpiperidino)cyclohexanol (AH 5183) on the veratridine-induced increase in acetylcholine synthesis by rat hippocampal tissue.

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by L-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of L-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergent-soluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

Immunocytochemical and pharmacological evidence for an intrinsic cholinomimetic system modulating prolactin and growth hormone release in rat pituitary.

Pituitary cells were cultured as three-dimensional reaggregates in serum-free chemically defined medium supplemented with different concentrations of dexamethasone. Immunostaining of the cells using a polyclonal antiserum and three monoclonal antibodies raised against choline acetyl transferase (CAT), revealed the presence of CAT immunoreactivity in 4-10% of anterior pituitary cells depending on the antibody used. CAT immunoreactivity was also found in freshly dispersed anterior pituitary cells. CAT-immunoreactive cells could be enriched on BSA and Percoll gradients and codistributed with ACTH-immunoreactive cells in these gradients. Perifusion of the aggregates with the potent muscarinic receptor antagonist atropine (Atr) resulted in a dose-dependent (0.1-100 nM) increase in both basal PRL and GH secretion; the response was dependent on the dexamethasone concentration in the culture medium. A similar response to Atr was observed in organ-cultured pituitaries. The specificity of the Atr effect was supported by the findings that the potent and highly specific muscarinic receptor blocker dexetimide showed a similar action, whereas its inactive enantiomer levetimide and the nicotinic receptor blocker hexamethonium failed to do so. Two other muscarinic antagonists, benzatropine and pirenzepine, showed a dose-dependent hormone-releasing action similar to that of Atr, but were less potent than the latter. Pirenzepine was only effective at high molar concentrations, suggesting that an M2 muscarinic receptor subtype was mediating the present phenomenon. Atr also potentiated GH release stimulated by the beta-adrenergic agonist isoproterenol and PRL release stimulated by vasoactive intestinal peptide, but had no effect on GRF-stimulated GH release. The choline uptake blocker hemicholinium abolished the effect of Atr on GH and PRL release. These data suggest that certain pituitary cells can express CAT activity and that these cells exert a tonic inhibitory activity on GH and PRL release which is mediated by a cholinomimetic substance, possibly acetylcholine, through a muscarinic receptor.

Approaches to protection against nerve agent poisoning. (Naphthylvinyl)pyridine derivatives as potential antidotes.

Analogues of the potent inhibitor of choline acetyltransferase (CAT) (E)-4-(1-naphthylvinyl)pyridine methiodide were synthesized and evaluated for their ability to inhibit CAT and protect against nerve agent intoxication. Several compounds, notably (E)-1-(2-hydroxyethyl)-(1-naphthylvinyl)pyridinium bromide (3), (E)-1-methyl-4-(1-naphthylvinyl)-1,2,3,6-tetrahydropyridine hydrochloride (22), and (E)-1-methyl-4-(1-naphthylvinyl)piperidine hydrochloride (23), were found to afford significant protection against sarin in the mouse and against soman in the guinea pig. However, protection was apparently not related to CAT inhibition. Compound 23, our most effective compound in protecting against nerve agent, was without CAT inhibitory activity. Compound 22, which proved to be a potent CAT inhibitor, most likely owed this activity to being dehydrogenated back to the pyridinium quaternary salt by oxidative enzymes. Several of the (naphthylvinyl)pyridine quaternary salts, but not their tertiary amine analogues, were found to be effective in slowing the rate of aging of soman-inhibited acetylcholinesterase. Ability to slow the rate of aging was enhanced by introduction of methoxy substituents on the aryl moiety whereas the aging rate was actually accelerated by chloro substituents. To date, our most effective compound in slowing the rate of aging, (E)-4-[(4-methoxy-1-naphthyl)vinyl]pyridine methochloride (6), did not provide significant protection against soman in the mouse.

Acetylcholine levels in the spinal cord after atropine, diisopropylfluorophosphate and naphthylvinylpyridine.

The decay of ACh levels from the death of the rat to longer time intervals has been estimated in the rat cervical spinal cord. The ACh levels at the beginning of the postmortal decay have been evaluated by extrapolation to t = 0 of the linear regressions log ACh concentrations vs time (t) from the rat decapitation. Atropine, diisopropylfluorophosphate (DFP) and naphthylvinylpyridine (NVP) caused a reduction of the ACh levels evaluated before the beginning of the postmortal decay. The levels of ACh at stabilization of the postmortal decay were increased by atropine and DFP. These effects may be correlated to modifications caused by the studied drugs on the ACh synthesis, release and destruction.

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: an intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)

4-(1-Naphthylvinyl)pyridine decreases brain acetylcholine in vivo, but does not alter the level of acetyl-CoA.

The effects of intraperitoneally administered 4-(1-naphthylvinyl)pyridine (NVP; 200 mg/kg) on the concentrations of acetylcholine (ACh), choline (Ch), and acetyl-CoA (AcCoA) in rat striatum, cortex, hippocampus, and cerebellum were investigated. Twenty minutes after treatment, the content of ACh was significantly diminished, whereas that of Ch was increased. In response to stress (swimming for 20 min), these changes were enhanced. However, the AcCoA content did not change in any of the brain regions. It is thus very likely that the decrease of brain ACh concentration induced by NVP is due to the drug's effect on choline acetyltransferase (ChAT) and/or the reduction of the high-affinity Ch uptake, and not on the availability of AcCoA. Presumably, the pharmacologically diminished activity of ChAT may become the rate-limiting factor in the maintenance of ACh levels in cholinergic neurons.

Development of choline acetyltransferase (CAT) in the sympathetic innervation of rat sweat glands.

It has been postulated that the developing sympathetic innervation of rat eccrine sweat glands changes from adrenergic to cholinergic under the influence of its target. In agreement with previous evidence that the sympathetic innervation of adult rat sweat glands is cholinergic, we found that choline acetyltransferase (CAT)-immunoreactive nerve fibers are present in adult glands, and that gland-rich chunks of adult footpads contain CAT enzyme activity. We were therefore interested in determining when CAT activity is first expressed in the developing gland innervation. Low levels of acetylating activity were observed in rat footpads as early as postnatal day 4, when sympathetic fibers first contact the glands. A greater than fourfold increase in CAT specific activity occurred between postnatal days 11 and 21. Neonatal treatment of rats with the adrenergic neurotoxin 6-hydroxydopamine (6-OHDA) eliminated most of the CAT activity in 14 and 19 d footpads. In contrast, the acetylating activity observed prior to day 11 was unaffected by neonatal 6-OHDA treatment, and only slightly reduced by the selective CAT inhibitor, naphthylvinylpyridine. These results indicate that the sympathetic fibers that innervate rat sweat glands do not acquire detectable levels of CAT activity until a full week after they contact the glands.

Structural correlations of choline acetyltransferase inhibitors: trans-N-(carboxymethyl)-4-(beta-1-naphthylvinyl)pyridinium bromide and cis-N-(2-aminoethyl)-4-(beta-1-naphthylvinyl)-3-methylpyridinium bromide hydrobromide.

This paper correlates the X-ray structures of two 4-(beta-1-naphthylvinyl)pyridine analogues (one cis and one trans) with chemical and biological activity data for this class of cholineacetylase inhibitors. Our results suggest that one of the two proposed mechanisms for inhibition by this class of compounds better describes their efficacy. Previous arguments about coplanarity of the aromatic rings and nucleophilicty across the vinyl linkage need to be modified. Quantum calculations are also included and substantiate previous suggestions about the charge distribution across the vinyl linkages. An alternate new mechanism of inhibition is proposed to encompass the published data and more recent results discussed in this paper.

Multiple forms of choline-O-acetyltransferase in mouse and rat brain: solubilization and characterization.

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.

Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase.

N-(10-carboxy)decamethylene-4(1-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a ligand for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quaternization of naphthylvinylpyridine with 11-Br-undecanoic acid methyl ester to obtain N-(10-carbomethoxy)decamethylene-4-(1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C11-NVP+) had a potency comparable to that of N-methyl-4(1-naphthylvinyl) pyridinium iodide (C1-NVP+) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C11-NVP+ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-C11-NVP+ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a "high" or a "low" sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I50 with C1-NVP+ = 0.57 microM or 5.2 microM). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the affinity column.

The effect of choline acetyltransferase inhibition on acetylcholine synthesis and release in term human placenta.

The synthesis and release of acetylcholine (ACh) was studied in term human placental villous mince in vitro. During a 140-min incubation the placental tissue synthesized ACh at a rate of 2.59 nmol/g/min and released ACh into the medium at a rate of 0.78 nmol/g/min. Consequently there was an increase in tissue levels of ACh from an initial value of 83 to 321 nmol/g. Inhibition of choline acetyltransferase by 2-benzoylethyl trimethylammonium or 4-(1-naphthylvinyl)pyridine depressed the synthesis of ACh by over 75% and blocked the increase in tissue levels of ACh. The IC50 values for the inhibition of ACh synthesis and decrease in tissue levels were close to the IC50 values determined for inhibition of choline acetyltransferase in situ. Neither 2-benzoylethyl trimethylammonium nor 4-(1-naphthylvinyl)pyridine caused a significant effect on ACh release. 2-benzoylethyl trimethylammonium and 4-(1-naphthylvinyl)pyridine were quite effective in inhibiting the uptake of the neutral amino acid, alpha-aminoisobutyric acid, into the tissue. The inhibition of alpha-aminoisobutyric acid uptake paralleled the inhibition of ACh synthesis. These results support the hypothesis of an association between placental cholinergic activity and amino acid transport in the human placenta.

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals.

The effects of hemicholinium-3 (HC-3) or 4-(l-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 micrograms HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR), against soman was increased excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.